Biodiesel is methyl or ethyl esters of fatty acids and efficient alternative to non renewable petroleum fuel with superior combustion qualities. It is produced by the transesterification of vegetable and oil produced from microbial oleaginous biomass such as Algae and Yeast. The chemical nature of neutral lipids, triacyl glycerols(TAG) and free fatty acids(FFA), present in oleaginous biomass is similar to those present in vegetable oil. Algae range from small, single-celled organisms to multi-cellular organisms. Algae are comprised of neutral lipids, glyco lipids, phospho lipids, proteins, vitamins etc. The neutral lipid fraction includes: triacyl glycerol( TAGs), free fatty acid(FFAs), sterols, wax and sterol esters, metals, hydrocarbons and free alcohols. Algal oil neutral lipids (TAG, FFA) are potential source of biodiesel produced by photosynthetic organism /strains utilizing light, water and carbon dioxide. Many micro algae have the ability to produce substantial amounts of 20–50% of dry cell weight of TAG as a storage lipid on account of photo-syntheses. It has been believed as one of good candidates for biodiesel production based on higher photosynthetic efficiency, higher biomass production and faster growth compared to other energy crops of vegetable oil. The nature of fatty acid present in algae or yeast biomass is dependent on the types of microbe/stains and culture conditions including feed stocks for their cultivation. Basically, the fatty acid composition of neutral lipids (Glycerides) has the same profile resembling vegetable oil or fish oil (C12 to C20/24) including unsaturated and PUFAs C16:1, C16:2, C18:1, C18:2 and C18:3, C20:5 and C22:6. Chemical Solvent extraction methods in combination with milling are applied to extract neutral lipids. Chemical Methods involves extraction by solvents such as Hexane/cyclohexane, Chloroform, methanol, Isopropanol and their mixtures by use of Soxhlet, Accelerated Solvent Extraction (ASE), Sonication and Supper Fluid Critical (SFC) methods. These are expensive due to energy and capital extensive and add enormous cost to the end product biodiesel.

Analytical Strategies

Analytical techniques such as chromatographic methods (GC, HPLC, GPC, SFC) spectroscopic (NMR, IR, MS, UV/Fluorescence) and Atomic spectrometry(XRF, ICAP/ICPMS, XRD, TEM, SEM) are being used extensively for compositional analyses and monitoring the production, synthesis, quality of feed stocks and conversion to bio diesel. The biodiesel potential of harvested dry biomass or algal oil is assessed by the yield of neutral lipids and composition of fatty acid comprising TAG and FFA. High-resolution Nuclear Magnetic Resonance( NMR) spectroscopic techniques(ID & 2D) due to their inherent sensitivity, high resolution and multinuclear capability offer many advantages over existing analytical methods due to ease of direct, rapid, simultaneous, and nondestructive analyses in the multi-component system of fat, oil and biodiesel obtained from various feed stocks.

The most common direct methods for quantitative analysis of neutral lipids from dry mass are solvent extraction for gravimetric lipid quantification, fat staining by fluorescence microscopy using a lipid stain such as Nile Red and solid state and Time domain Nuclear Magnetic Resonance (TDNMR). Among all the spectroscopic methods, time domain nuclear magnetic resonance (TD-NMR) or low resolution or low field, has been the most popular solution for quality control and cultivation of algae with regard to neutral lipids in the industrial sector. The fat staining methods Nile Red and spectroscopic TDNMR and FTIR are qualitative and sometimes are specific to the types of oleaginous microbial strains and do not provide detailed compositional analyses including fatty acid profile. These parameters are essentially required to assess the potential and feasibility of production and quality of bio diesel from a particular feed stock. The data will also facilitate bio diesel technology developer to select suitable feed stock, process and appropriate catalyst.

As described above, the detailed compositional analyses of Whole Algae mass are carried out on the Algal oils obtained by solvent extraction. The solvent extraction methods require extensive usage of solvents, samples, equipments and also energy extensive as solvents are to be removed before application for elaborate analytical studies. It has been shown by us that the extracts obtained by various extractions methods are not pure and besides neutral lipids; proteins, glycolipids, sterols, phospholipids are extracted in large quantities as evidenced by the analyses of these extracts by combination of spectroscopic and chromatographic techniques. The Algal extracts have the tendency to degradation, hydrolyses and oxidation during extraction and evaporation while removing the solvents. Thus true representative extracts are not generated for subjecting to analyses by various analytical techniques for the real composition of Algae under studies. Since the process of extraction is time consuming and if immediate analyses is required in order to monitor the total lipid content and assess the quality of the fresh algae harvested; a rapid, reliable, convenient and less energy intensive extraction and analytical method is required. In order to provide rapid, convenient and quality detailed analyses, a novel concept has been successfully conceived for the direct analyses of dried whole algae sample in NMR tube. The method is based on ultrasonic extraction of neutral lipids from dry algae mass in NMR tube for obtaining an appropriate amount of extract so that quick analytical information is obtained. Rapid determination of lipid content in microalgae cell is important for monitoring and optimization of harvesting process for enhancement of the neutral lipid content.

The basic theory of concept is the extraction of total lipids in deuterated chloroform( 0.6ml, CDCl3) from the whole solid algae mass directly in NMR tube by ultrasonication in a sonication bath at room temperature. Rate of extraction and efficiency of neutral lipid extraction can be enhanced by optimizing the power of Sonication. The time of ultrasonic extraction is 5 to 10 minutes depending upon the power of Sonication waves, which can be applied between 50 to 700 W. The amount of solid biomass required is 15 to 30 mg, being optimum of 20 mg for 500 and 600MHz NMR instruments for achieving better spectral sensitivity and resolution. The stable solution so obtained is less prone to oxidation. Immediately, NMR tube is inserted into the probe of NMR instrument and both ID and 2D NMR spectra are obtained and interpreted for nature of components present. The total lipid content and FFA profile Iodine value etc. can be estimated by using internal standards. Since NMR is a non destructive technique, the solution is filtered after the recordings of NMR and further analyzed by GC, HPLC, GPC, IR, ICAP, ICPMS etc. for detailed component and metal analyses. The concept has been applied successfully to samples containing lipid content of 5 to 40% and results better than those of conventional solvent extraction methods has been obtained. The analytical strategies developed are not energy and solvent intensive and Concept of Green Chemistry is evolved for rapid and convenient multinuclei and multi dimensional ID & 2DNMR analyses. The cost effective diverse multiple analytical techniques approach with minimum quantity of sample and solvent has been adopted. The rapid determination of lipid content in microalgae cell has been achieved, which is important for monitoring and optimization of harvesting process for enhancement of the neutral lipid content. Ultra Sonication extraction at room temperature may provide the choice in favor of neutral lipids by varying the power of Sonication waves.